Microbiological quality assessment of Clarias gariepinus, Bagrus bajad, and Pangasianodon hypophthalmus fillets

In this study, 80 catfish fillets were randomly collected from Egyptian local markets and retailers. The samples included 20 African catfish (Clarias gariepinus), 20 bayad (Bagrus bajad), and 40 pangasius catfish (Pangasianodon hypophthalmus) fillets. Pangasianodon hypophthalmus fillet samples were divided into 20 white basa and 20 red basa fillets. We conducted a microbiological analysis of catfish fillet samples, evaluating mesophilic aerobic bacteria, psychrophilic aerobic bacteria, H2S-producing bacteria, Staphylococcus spp., Enterobacteriaceae, Coliforms, and fecal Coliform counts. Additionally, we identified the existence of Salmonella spp., Vibrio spp., Yersinia spp., Escherichia spp., Aeromonas spp., and Pseudomonas spp. in the catfish fillet samples. In our study, the psychrophilic bacterial counts in Bagrus bajad (5.21 log CFU/g) were found to be higher compared to the counts in Clarias gariepinus (4.31 log CFU/g) and Pangasianodon hypophthalmus (3.89–4.7 log CFU/g). The fecal Coliform in Clarias gariepinus fillets was significantly higher than in other catfish fillets. We isolated Escherichia coli, Escherichia fergusonii, Aeromonas hydrophila, and Pseudomonas luteola from the catfish fillets, while no Salmonella spp., Vibrio spp., or Yersinia spp. were detected. These isolates were identified using 16S rRNA sequencing and phylogenetic analysis. Furthermore, ten Escherichia spp. were serologically identified, revealing that O26 and O78 were the most commonly occurring serotypes. This study highlights the microbiological analysis conducted on catfish fillets and concludes that the fillet samples from these catfish were of superior quality and deemed acceptable for human consumption.


Sample size calculation
The determination of the sample size for this study followed the formula for an unknown population, as outlined by Kothari 26 : n = Z 2 SD 2 /e 2 , In this calculation, where Z represents the value of the standard variate (1.96) at a 95% confidence level, SD denotes the standard deviation of the population derived from the trial sample (0.11), and e stands for the tolerable sampling error or precision (0.05) within a 95% confidence interval.Subsequently, the sample size was computed as: Therefore, the sample size of each catfish fillet type was 19, making a total of 80 samples for the four types of fillets (African catfish fillets, Bayad fillets, white basa fillets and red basa fillets).

Samples collection
In total, 80 samples were collected.This included 20 African catfish (C.gariepinus) fillets weighing between 600 and 1000 g, 20 bayad (B.bajad) fillets weighing between 500 and 2500 g and 40 skinless-frozen pangasius catfish (P.hypophthalmus) fillets weighing between 600 and 800 g.These samples were obtained from fish markets and retailers in Egypt (Kafr El Sheikh, Alexandria and Beheira governorates) between 2021 and 2023.Pangasius catfish fillets on the Egyptian market can be divided into two grades: white basa fillets (20 samples) and red basa fillets (20 samples).All catfish fillet samples were transported in separate iceboxes filled with ice bags to the Department of Food Hygiene, Alexandria University.Microbiological analyses were performed immediately on the catfish fillet samples.

Enumeration of bacterial load
Upon arrival at the laboratory, sterile scalpels and tweezers were used to aseptically collect 25 g samples of fish fillet, which were then placed in a sterile stomacher bag.Next, 225 mL of sterile 0.1% peptone water (Difco, UK) was added to the bag, and the mixture was homogenized for 1 min using a Stomacher at the normal speed (1.96) 2 (0.11) 2 (0.05) 2 ≈ 19 samples (Stomacher lab-blender 400, Seward Medical, UK) according to ISO 6887-3:2017 27 .Subsequently, a tenfold serial dilution series was prepared, and the counts were determined using the pour plate technique according to ISO 6887-3:2017 27 .Each analysis was conducted twice in order to ensure accuracy.The number of viable microorganisms was then counted, calculated, and expressed as the logarithm of colony-forming units per gram (log CFU/g).Plate Count Agar (PCA) is a widely employed solid culture medium for enumeration the viable bacterial population in a sample.It provides a nutrient-rich environment that supports the growth of a wide range of bacteria.PCA contains a combination of peptones, yeast extract, and agar according to ISO 4833-1:2013 28 .The mesophilic aerobic count and psychrophilic aerobic bacteria were determined using a Plate Count Agar (PCA, Oxoid), then incubated at 30 °C for 48 h and at 7 °C for 7-10 days, respectively according to according to ISO 6887-3:2017 27 ; ISO 4833-1:2013 28 .The hydrogen sulfide producing bacteria were enumerated using iron agar (14 g agar, 3 g beef extract, 20 g peptone, 5 g sodium chloride, 3 g yeast extract, 0.6 g L-Cysteine, 0.3 g Sodium thiosulfate and 0.3 g Ferric citrate per 1L autoclaved distilled water) according to Gram et al. 29 .Staphylococcus spp.and Staphylococcus aureus counts using Baird-Parker agar medium according to ISO 6888:2021 30 .Enterobacteriaceae counts were enumerated using Violet red bile glucose (VRBG) agar and incubated at 37 °C for 24 h according to ISO 21,528:2017 31

Isolation of pathogenic bacteria
Isolation of E. coli was conducted according to ISO 16,649:2018 36 .Approximately 1 g of homogenized fish fillets was mixed with 9 mL of modified Tryptone Soya Broth (mTSB, HiMedia).The samples were thoroughly mixed and left to incubate overnight at a temperature of 41 °C.Following selective enrichment, 50 µL of the resulting mixture were spread onto MacConkey agar (HiMedia) plates to isolate E. coli bacteria, and the plates were incubated aerobically at 37 °C for 24 h.The plates were then examined for the presence of E. coli growth, characterized by pink colonies indicating lactose fermentation.A single, isolated colony exhibiting these characteristics was chosen and transferred to Eosin Methylene Blue agar (EMB, HiMedia) to observe the formation of a metallic sheen.At the same time, another colony displaying similar characteristics was subjected to gram staining according to ISO 16,649:2018 36 .
Isolation of Salmonella spp. was performed according to ISO 6579:2017 37 .Briefly, 225 mL of buffered peptone water was inoculated with 25 g fish fillets and incubated at 37 °C for 18 h (pre-enrichment in non-selective liquid medium), then inoculate 1 mL of the above-mentioned broth into 9 mL Rappaport-Vassiliadis medium with soya (RVS broth, Oxoid) and finally plating out on Xylose Lysine Deoxycholate agar (XLD, Oxoid) and Salmonella Shigella agar (SS, Oxoid), incubated at 37 °C for 24 h according to ISO 6579:2017 37 .
Vibrio spp.were performed according to ISO 21,872:2017 39 .Isolation of Vibrio spp. was starting from primary enrichment medium alkaline saline peptone water (ASPW, Oxoid), followed by incubation at 37 °C for 6 h, then streaked on thiosulfate citrate bile and sucrose agar (TCBS, Oxoid) and incubated at 37 °C for 24 h according to ISO 21,872:2017 39 .
Representative colonies were selected from the plate count agar (PCA, Oxoid) after incubation at 7°C for 7-10 days.Selected colonies were streaked onto Rimler-Shotts (RS, HiMedia) agar supplemented with Novobiocin (HiMedia), and Pseudomonas agar base enriched with Cetrimide, Fucidin, and Cephalosporin (Pseudomonas CFC agar, Oxoid) and then placed in an incubator at 25 °C for a period of 24 to 72 h.Typical Aeromonas spp.produce greenish-yellow to yellow colonies, or yellow colonies with black centers (H 2 S producing bacteria) on RS agar, while typical Pseudomonas spp.produce blue-green colonies on Pseudomonas CFC agar.

Phenotypic characterization of isolates
The presumptive identification of isolates was accomplished by assessing their phenotypical characteristics according to the criteria described by Bergey 40 .Biochemical characterization of the isolates using commercial miniaturized API-20E (Biomérieux, France) were performed according to the manufacturer's instructions.Confirmed isolates were maintained until further use at − 20°C in nutrient broth containing 16% glycerol.

Serological identification of Escherichia spp. isolates
A total of ten isolates of E. coli and E. fergusonii, which were identified based on their phenotypic characteristics, underwent serological identification according to Ewing 41 .

Genotypic characterization, 16S rRNA sequencing and Phylogenetic analysis
Four bacterial isolates were selected for sequencing studies based on their morphological and biochemical characteristics.The selected bacterial isolates were Aeromonas hydrophila, isolated from Bagrus bajad, Pseudomonas luteola, isolated from Pangasianodon hypophthalmus as well as E. coli, and E. fergusonii, isolated from Clarias gariepinus.The DNA extraction of these isolates was performed using QIAamp DNA Kits (Qiagen, USA) following the instructions provided by the manufacturer.The DNA was maintained until further use at − 20 °C.The genotypic identification of isolates was confirmed by employing universal 16S rRNA gene primers (Forward: 5′-AGA GTT TGA TCC TGG CTC AG-3′) (Reverse: 5′-GGT TAC CTT GTT ACG ACT T-3′) 42 .The 16S rRNA gene was amplified using 50 µL reaction volume in Maxima Hot Start PCR Master Mix (ThermoFisher, USA), per the manufacturer's instructions.The PCR procedure was set with an initial denaturation step at 95 °C for 10 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 65 °C for 60 s, and extension at 72 °C for

Statistical analyses
Statistical analyses were performed via the R program (R 4.3.1) 47.The heteroscedasticity of variances and the normality of residuals was calculated using the Levene's and Shapiro-Wilk tests.Microbiological assessment data of catfish fillets was presented as the mean ± SEM (Standard error of mean).Microbiological data were computed by one way analysis of variance (ANOVA) and followed by Tukey's post hoc test for multiple comparison between groups.The significance level was set at a probability value of less than 0.05 (p ˂ 0.05).

Qualitative microbiological analyses
In the present study, concerning Salmonella spp., Yersinia spp.and Vibrio spp., no colony was isolated from our catfish fillet samples.On the other hand, Escherichia spp. was detected in five African catfish fillet samples, three bayad fillet samples and two pangasius catfish fillet samples (Table 2).The incidence of Escherichia spp.isolated from catfish fillets were 25%, 15%, 5% and 5% for African catfish fillet samples, bayad fillet samples and white basa fillet samples and red basa fillet samples.In the other hand, the incidence of Aeromonas spp.isolated from catfish fillets were 25%, 20%, 5% and 20% for African catfish fillet samples, bayad fillet samples, white basa fillet samples and red basa fillet samples.Additionally, the incidence of Pseudomonas spp.isolated from pangasius catfish fillets were 5% and 15% for white basa fillet samples and red basa fillet samples (Table 2).Table 1.Bacterial load of examined African catfish (Clarias gariepinus), bayad (Bagrus bajad) and pangasius catfish (Pangasianodon hypophthalmus) fillets.Data represented as mean of 20 samples (duplicate) ± SEM-Standard error of the mean, rows with distinct superscripts exhibited statistically significant differences, as determined by ANOVA and Tukey's post-hoc test, with a significance level of p ≤ 0.05.Pangasius catfish fillets can be divided into two grades: white basa fillets (grade I, white colour) and red basa fillets (grade II, pale pink to red colour).The number between parenthesis refers to number of positive samples out of 20 samples.4. In this study, we isolated E. coli serotype O26 from African catfish, bayad, and pangasius catfish fillets, while E. fergusonii serotype O78 was found in two African catfish fillet samples.Following 16S rRNA sequencing analysis, the phenotypically and serologically identified Escherichia isolates were assigned to E. coli and E. fergusonii.Additionally, the construction of the phylogenetic tree utilizes 16S rRNA gene sequences, with length of 1432 bp (E.coli, MT845092) and 1386 bp (E.fergusonii, MT844056) is shown in Fig. 3. Compared to the sequences available in GenBank, E. fergusonii 16S rRNA gene (MT844056) expressed a 99.5% homology with the 16S rRNA sequence of E. fergusonii (JQ838153 and MH040100), while E. coli 16S rRNA gene (MT845092) expressed 99.9% homology with the 16S rRNA sequence of E. coli (KT260583 and MF754138).This study reports the first isolation of E. fergusonii from African catfish fillet samples.

Discussion
In this study, we evaluate the levels of spoilage and pathogenic microorganisms in catfish fillet samples.Most of the evaluated microbiological properties are within the permissible limits set by International Commission on Microbiological Specification for Food 48 .Psychrophilic aerobic bacteria of bayad fillets (> 5 log CFU g −1 ) were significantly higher than African catfish fillets (> 4 log CFU g −1 ) and pangasius catfish fillets (white basa fillets only: ≤ 4 log CFU g −1 ).Dambrosio et al. 14 indicated that the average psychrophilic aerobic bacteria count in fillet samples of P. hypophthalmus, acquired from an Italian trade import services company, was 4.44 log CFU g -1 and these results were comparable with our results.Moreover, the counts of aerobic psychrotrophic microorganisms found in pangasius catfish varied from 4.6 to 5.9 log CFU g -121 .Nevertheless, although CFU g -1 obtained in this study was moderately high, it did not exceed the acceptable permissible limit for total bacterial load (5.5-7.0 log CFU g -1 ) for fresh and frozen fish, as established by International Commission on Microbiological Specification for Food 48 .The elevated levels of psychrophilic aerobic bacteria found in bayad fillets may be attributed to the preservation method commonly used for bayad in Egyptian markets, which involves storing it with an equal amount of crushed ice.
Staphylococcus aureus does not naturally inhabit the microbiota of fish.Consequently, its occurrence in fish is possibly linked to unsanitary practices during handling by fish handlers, processors, or sellers, as well as potential cross-contamination throughout handling, transportation, storage, and processing, stemming from the presence of this pathogen in the microbiome of most humans [49][50][51] .Although Staphylococcus spp.count in our pangasius catfish fillet samples were higher than 4 log CFU g -1 , Staphylococcus aureus was estimated to be 1.33 to 1.51 log CFU g -1 .These moderately high levels suggested that the product contamination is possibly linked to unsanitary practices during handling, processing, selling, and storage etc. [50][51][52] .Tong Thi et al. 21indicated that the detection of Staphylococcus aureus on the hands of food operators during fish processing, especially in the packaging area, was deemed indicative of inadequate personal hygienic practices.Lower Staphylococcus aureus level (1.14 log CFU g -1 ) in pangasius catfish fillets was previously reported by Dambrosio et al. 14 .Our findings were lower than the acceptable permissible limit of Staphylococcus aureus (˂ 3 log CFU g -1 ) in fish fillets set by Egyptian Organization for Standardization and Quality 53 .
The quantification of specific spoilage organisms (SSOs) on iron agar is a more reliable microbial measure of fish freshness; SSOs are responsible for the deterioration of fish and seafood 17,18 .Hydrogen sulfide producing bacterial counts in pangasius catfish fillets (red basa fillets; 2.91 log CFU g −1 ) revealed higher counts than African catfish fillets (2.65 log CFU g −1 ), bayad fillets (2.33 log CFU g −1 ) and pangasius catfish fillets (white basa fillets; 2.57 log CFU g −1 ).The Enterobacteriaceae count in pangasius catfish fillets samples was 2.86 to 3.01 log CFU g -1 for white basa and red basa, which is higher than the Enterobacteriaceae count in pangasius catfish fillets samples Table 2. Pathogenic bacterial isolates retrieved from African catfish (Clarias gariepinus), bayad (Bagrus bajad) and pangasius catfish (Pangasianodon hypophthalmus) fillets.ND-Not detected..Enterobacteriaceae and Coliform levels in fish fillets are an indicator of general bacteriological conditions, and an index for the presence of pathogenic enteric organisms 54 .Mossel and Tamminga 55 adopted a reference value (3 log CFU g −1 ) for Enterobacteriaceae in fish fillets.In the present study, the mesophilic aerobic counts, psychrophilic aerobic bacteria, Staphylococcus aureus and Enterobacteriaceae counts of catfish fillets were within the acceptable permissible limit set by Egyptian Organization for Standardization and Quality 53 ; International Commission on Microbiological Specification for Food 48 .Fecal Coliforms are a group of bacteria most commonly used as pollution indicators in food and water and easily affected by freezing storage 56 .The fecal Coliform count in African catfish fillet (1.27 log MPN g −1 ) was significantly higher than bayad fillets (0.91 log MPN g −1 ) and pangasius catfish fillets (0.50-0.56 log MPN g −1 ).These levels were lower than the upper acceptable permissible limits of fecal Coliform for fish fillets set by Egyptian Organization for Standardization and Quality 53 ; International Commission on Microbiological Specification for Food 48 .Comparable results were previously documented by Budiati et al. 57 , who observed that the fecal Coliform content for catfish ranged between 0.48 and 1.63 log MPN g -1 .The lower levels of fecal Coliform found in white basa (0.50 log MPN g -1 ), and red basa (0.56 log MPN g -1 ) fillets may be attributed to the freezing preservation method commonly used for basa fillets in Egyptian markets.Boyd and Tanner 58 reported that the high organic matter, poor water quality, inferior feed quality and high stocking density of catfish in ponds could be associated Table 3. Phenotypic characterization of isolates retrieved from African catfish (Clarias gariepinus), bayad (Bagrus bajad) and pangasius catfish (Pangasianodon hypophthalmus) fillets.+ : Positive, -: Negative, -/ + : Most isolates are negative, + /-: Most isolates are positive, A/Ag: Acid slant (yellow) and acid butt (yellow) with gases, K/A: Alkaline slant (red) and acid butt (yellow), K/K: No change in slant (red) and alkaline butt (red).O/F: Oxidation fermentation positive, O: Oxidation positive.Number in parenthesis indicated number of isolates identified by phenotypic characterization.   suggested that the type of feed can influence the bacterial burden in fish.Utilizing chicken offals and spoiled eggs as fish feed may pose potential sources of bacterial contamination in both fish and aquatic environment.Enterobacteriaceae, Staphylococcus spp., and various other microorganisms may be present in the initial microbial population, primarily as contaminants 59 .No Salmonella spp. was detected in our fillet samples and comparable findings were previously documented by Dambrosio et al. 14 who did not detect Salmonella spp.from P. hypophthalmus fillet samples imported to Italy.Similar findings were reported regarding Vibrio spp.by Noseda et al. 60 , where they did not detect Vibrio spp. in P. hypophthalmus fillets.Nevertheless, in contradiction to our results, Tong Thi et al. 21detected V. cholerae (in 1/9 samples) of the pangasius catfish sampled at the filleting step.

Microorganism
Escherichia spp. is the predominant Coliform found in the intestinal flora of warm-blooded animals and is primarily linked to fecal contamination 60 .During the processing stage, high levels of E. coli were detected in the samples collected from hands and surfaces due to cross contamination from food contact surfaces (hands, cutting boards and knives) and fish fillets 21,60 .Moreover, the presence of Escherichia spp. in fish fillet might be attributed to the contamination of fishponds by livestock waste 61 .Yagoub 62 claimed that the fertilization of the fishpond using farm animal and poultry manure could be a source of E. coli in the fish samples.In contrast, low Escherichia spp. was detected in pangasius catfish fillets, suggesting that the freezing process had lethal effect on Escherichia spp.Fish fillets contamination by Escherichia spp.may be associated with contaminated water or through cross-contamination during washing, filleting, and trimming of the fish 21 .
The incidence of Aeromonas spp.isolated from catfish fillets were 25%, 20%, 5% and 20% for African catfish fillet samples, bayad fillet samples, white basa fillet samples and red basa fillet samples.According to Henin 63 & Ibrahim et al. 64 , the incidence of Aeromonas spp. in imported frozen fish, fresh catfish and freshwater fish was reported as 15.2%, 11.6% and 9.7%, respectively.Wong et al. 65 detected Aeromonas spp. in 10% of the frozen fish samples they examined.In contrast, Pseudomonas spp. was exclusively detected in samples of pangasius catfish fillet.Higher results were reported by Yagoub 62 who isolated Pseudomonas spp.from 62% of the examined fish samples and Rahmou 66 who isolated Pseudomonas spp.from 28% of the examined fish fillet samples.The specific spoilage organisms (SSOs) in the present study were Aeromonas spp.and Pseudomonas spp., these results agree with Viji et al. 67 .Previous studies mostly defined the SSOs in aerobically stored fish and fish products as Gramnegative psychrotrophic bacteria, including Pseudomonas spp., Aeromonas spp., Vibrio spp., and Shewanella spp. 18,22,68,69.Pseudomonads are one of the most significant spoilage organisms, as their rapid growth contributes to the breakdown of nitrogenous compounds, ultimately resulting in the deterioration of product 22 .
Aeromonas hydrophila (A.hydrophila) is ubiquitous in the aquatic environment and has been found in freshwater fish, including catfish and tilapia [70][71][72] .Pseudomonas luteola are Gram-negative, aerobic, oxidase-negative rods commonly found in aqueous environments, soil and plants 24,25 .Pseudomonas luteola is not a common pathogen in aquaculture; the first record of P. luteola infection in rainbow trout (Oncorhynchus mykiss) was reported by Altinok et al. 24 .This study reports the isolation of P. luteola in pangasius catfish fillets imported to Egypt.
E. coli is ubiquitous, as it naturally inhabits the intestines of warm-blooded animals without causing any symptoms, and it is extensively spread throughout the environment 73 .Thus, E. coli is a reliable indicator of fecal contamination, water pollution and mishandling 74,75 .E. fergusonii is an emerging opportunistic pathogen and is occasionally isolated from the intestinal contents of human and warm-blooded animals.Several studies have isolated E. fergusonii from mammals and birds with systemic or enteric infections 76,77 , whereas a few studies have isolated E. fergusonii from sewage, surface water, well water and cultured Egyptian Nile tilapia with signs of bacteremia 78,79 .E. fergusonii has been frequently identified in the fecal matter of cattle, poultry, goats, sheep, and horses exhibiting symptoms such as diarrhea, meningitis, mastitis, abortion, and septicemia 76,80 .The phenotypic analysis of E. fergusonii were nearly similar to isolates recovered from Egyptian Nile tilapia 78 except our isolates were positive for ADH (Arginine DiHydrolase) and negative for ONPG (ß-galactosidase).
E. coli serotype O26 was frequently isolated from African catfish, bayad, and pangasius catfish fillets, while E. fergusonii serotype O78 was found in two African catfish fillet samples.Certain serotypes (O26) found in African catfish were comparable to the predominant Escherichia serotypes identified in broiler chickens in Egypt 81,82 .E. coli is an extrinsic microorganism for fish environment, and it is not a part of fish flora.E coli might be introduced to fishponds through the traditional fertilization of fishponds using farm animals and poultry manure, which may harbor E. coli, E. fergusonii and other Enterobacteriaceae members 21,61,75 .

Conclusion
The present study aimed to assess the bacterial load and pathogenic bacteria in African catfish, bayad, and pangasius catfish fillets.Our findings indicate that all examined catfish fillets were deemed acceptable and safe for human consumption.No Salmonella spp., Yersinia spp., or Vibrio spp.were detected in any of the examined catfish fillets.E. coli serotype O26 was frequently isolated from African catfish, bayad, and pangasius catfish fillets, while E. fergusonii serotype O78 was found in two African catfish fillet samples.Furthermore, this study reports first isolation of E. fergusonii from African catfish fillets and Pseudomonas luteola from pangasius catfish fillets.The isolation of E. fergusonii from African catfish fillet samples highlights the need for more research on emerging pathogens and their prevalence in catfish production.To prevent contamination, recontamination, or the survival of biological hazards during handling, processing, distribution, and storage of catfish fillets, we highly recommend implementing Good Manufacturing Practices (GMP), Good Hygiene Practices (GHP), and a meticulously planned HACCP program.Continued surveillance and investigation of bacterial species can contribute to better understanding and management of risks associated with catfish fillets.Overall, the catfish industry, producers and consumers will benefit from using our data on microbiological quality assessment of the catfish fillets for stringent process control.

Figure 1 .
Figure1.Presents a phylogenetic tree of twenty identified Aeromonas spp.and Aeromonas hydrophila (Scf Ah1) isolate (Bagrus bajad), represented by a red rectangle.The tree was constructed using the neighborjoining method, and the aligned sequence had a length of 1485 bp.The bootstrap values (%) are displayed next to the clades, and the accession numbers are indicated before the strain names.The neighbor-joining tree was constructed using MEGA X, scale 0.05.

Figure 2 .
Figure 2. Presents a phylogenetic tree of twenty identified Pseudomonas spp.and Pseudomonas luteola (Pcf Pl1) isolate (Pangasianodon hypophthalmus), represented by a red rectangle.The tree was constructed using the neighbor-joining method, and the aligned sequence had a length of 1490 bp.The bootstrap values (%) are displayed next to the clades, and the accession numbers are indicated before the strain names.The neighborjoining tree was constructed using MEGA X, scale 0.05.

Figure 3 .
Figure 3. Presents a phylogenetic tree of twenty-eight identified Escherichia spp.and E. coli (Acf Ec2),and E. fergusonii (Acf Ef1) isolates (Clarias gariepinus), represented by red rectangles.The tree was constructed using the neighbor-joining method, with the aligned sequences having lengths of 1432 bp and 1386 bp, respectively.The bootstrap values (%) are displayed next to the clades, and the accession numbers are indicated before the strain names.The neighbor-joining tree was constructed using MEGA X, scale 0.05. https://doi.org/10.1038/s41598-024-62730-8 . The Most Probable Number (MPN) method was used for the enumeration of Coliform and fecal Coliform according to Feng et al. 32 ; ISO 7251:2005 33 ; ISO 4831:2006 34 ; Oblinger & Koburger 35 .
The results of the phenotypic analyses conducted on P. luteola and A. hydrophila in the current study are presented in Table3.The pure cultures of A. hydrophila and P. luteola were confirmed by sequencing 16S rRNA genes.Compared to the 1485 bp 16S rRNA gene of A. hydrophila (MT847230) expressed a 99.5% homology with the 16S rRNA sequence of A. hydrophila subsp.hydrophila(LC420139,KX012004andLC420130),whereas the sequences available in the GenBank, 1490 bp 16S rRNA gene of P. luteola (MT845202) expressed a 99.6% homology with the 16S rRNA sequence of P. luteola (KT728842, KY194220 and KY194291).The phylogenetic tree using 16S rRNA gene sequences of A. hydrophila and P. luteola is shown in Figs.1 and 2, respectively.The results of the phenotypic analyses conducted on E. coli and E. fergusonii in the current study are presented in Table3.The serological identification of eight E. coli and two E. fergusonii isolates are shown in Table Vol.:(0123456789) Scientific Reports | (2024) 14:13305 | https://doi.org/10.1038/s41598-024-62730-8www.nature.com/scientificreports/Phenotypic and genotypic characterization of isolates
with rising Coliforms and fecal Coliform loads in catfish fillets.Additionally, Budiati et al.